Cephalosporin compounds

ABSTRACT

7B-AMINO- AND ACYLAMIDOCEPH-3-EM-4-CARBOXYLIC ACIDS HAVING AT THE 3-POSITION A HALO-, FORMYLOXY-, ISOTHIOCYANATO- OR HALOACETOXYMETHYL GROUP AND SALTS AND ESTERS THEREOF. THESE COMPOUNDS ARE USEFUL AS STARTING COMPOUNDS FOR PREPARING 7B-ACYLAMIDOCEPH-3-EM-4-CARBOXYLIC ACIDS HAVING A GROUP AT THE 3-POSITION, OTHER THAN 3-ACETOXYMETHYL, BY REACTION WITH A NUCLEOPHILE. THE COMPOUNDS PREPARED HAVE MODIFIED ANTIBIOTIC ACTIVITY.

United States Patent Office 3,658,799 Patented Apr. 25, 1972 US. Cl.260-243 C 10 Claims This invention is concerned with improvement in orrelating to the production of analogues of cephalosporin C The compoundsin this specification are generally named with reference to cepham whichhas the structure (see J.A.C.S. 1962, 84, 3400).

7fi-acylamidoceph-3-em-4-carboxylic acids having a group at the3-position, other than 3-acetoxymethyl, are generally of interest inthat they possess modified antibiotic activity, as compared with thecorresponding 3-acetoxymethyl-7p-acylamidoceph-3-em-4 carboxylic acids,for exampleN-[7,8-(2'-thienylacetamido)ceph-3-em-3-ylmethyl]pyridinium-4-carboxylatehaving the accepted name cephaloridine possesses advantageous antibioticactivity as compared with 3-acetoxymethy1-73-(2'-thienylacetamido)cept-3-em-4-carboxylic acid.

British Pat. No. 1,012,943 describes and claims the direct displacementof the acyloxy group of the 3-acyloxymethyl substituent of suchceph-3-em-4-carboxylic acids by certain compounds of a nucleophilicnature. This type of process, however, is unsuccesful with somenucleophiles and in other cases low yields are obtained. Reaction withsulphur-containing nucleophiles often proceeds in good yields, butfactors such as the low solubility of some of the products anddeficiencies in activity against gram-negative organisms, render theproducts less useful as antibiotics. British Pat. No. 912,541 describesand claims the direct replacement of the acetoxy group of cephalosporinC compounds by compounds such as pyridine to form cephalosporin Ccompounds. This method leads to low yields of the desired 0,, compounds.

We have now discovered that a wider range of nucleophiles may be usedfor substitution reactions at the exocyclic methylene group if the3-acyloxymethyl group is first hydrolysed to the 3-hydroxymethyl groupwhich is then reesterified (if necessary after protective esterificationof the 4-carboxy group) with certain acids hereinafter defined, and thenallowed to react with the nucleophile. The process provides a usefulalternative to the method of said British Pats. Nos. 1,012,943 and912,541, furthermore, the method enables one to prepare cephalosporinanalogues diflicultly accessible by direct displacement of the acetoxygroup.

According to the present invention therefore we provide a process forthe preparation of a cephalosporin analogue which includes the step ofreacting a ceph-3-em- 4-carboxylic acid, or a derivative thereof, havingthe group RHN- at the 7B-position, wherein R represents a hydrogen atomor a carboxylic acyl group, said ceph-3- ern-4-carboxylic acid orderivative having the group at the 3-position (so that theceph-3-em-4-carboxylic acid or derivative used as starting material hasthe formula where Q is the remainder of the cephalosporin molecule) suchthat the acid HX has a pKa of not more than 4.0, preferably not morethan 3.5, and X does not contain a sulphur atom linked to the hydrogenatom, with a compound having a nucleophilic atom, e.g., a carbon,nitrogen, oxygen or sulphur atom, under conditions effective to displacethe group X by the nucleophile.

Where R is a hydrogen atom it may first be protected, e.g. by acylation,with a group which is subsequently removed. Alternatively, the hydrogenatom may be left unprotected in which event it may be involved insubsequent reactions. In general we prefer to use compounds in which Ris an acyl group.

The pKa values refer to aqueous solution at 25 C.

The specific classes of compounds having nucleophilic atoms will bereferred to for convenience as carbon, nitrogen, oxygen or sulphurnucleophiles in the followmg.

The group X is preferably one of three main types, depending whether ahalogen atom, oxygen atom or nitrogen atom is directly attached to theQCH -group (where Q is as hereinbefore defined); these three' types ofgroups will be discussed in turn under the respective headings:halogens; oxygen leaving-groups; and nitrogen leavinggroups.

HALOGENS (X=Cl, Br, or I) When X represents a halogen atom, we havefound that the 4-carboxy group may or may not be esterified. Sinceesters are generally without substantial antibiotic activity, andmoreover since the corresponding isomeric ceph-Z-em- 4-carboxylic acidsare also generally without substantial antibiotic activity, it isdesirable to use as esterifying group a group which can be readilyintroduced and removed without appreciable A' A isomerisation (which isknown to be likely to occur in such reactions). Preferred esterifyinggroups include the diphenylmethyl, the B,B,B-trichloroethyl and t-butylgroups; the diphenylmethyl group can be readily introduced by means ofdiphenyldiazomethane, and removed e.g. by means of a mixture oftrifluoroacetic acid and anisole at room temperature whilst the tSJiyS-trichloroethyl group may be removed by means of zinc and aceticacid.

The 3-CH X compounds, where X represents C1 or Br, can be prepared fromthe corresponding 3-CH OH compounds by standard methods for thereplacement of -0H by C1 or Br. For example, the 3-CH Cl compounds canbe prepared from the corresponding 3-CH OH compounds by reaction withthionyl chloride, acid chlorides such as N,N-dialkylorN,N-diarylchlorosulphinamides, e.g. N,N- dimethylchlorosulphinamides, oralkyl chlorosulphites. The 3-CH Br compounds can be similarly preparedby reaction of the corresponding bromo-compounds with the 3-CH OHcompounds or by reaction of the latter with PBr and pyridine. The 3-CH Icompounds may be prepared from the corresponding chlorides and bromidese.g. by reaction with an alkali metal iodide.

The 3-CH OH compounds can be prepared for example by hydrolysis of thecorresponding 3-CH .O.COCH

compounds. The hydrolysis is preferably eflFected enzymatically, using,for example, wheat germ esterase or an esterase of the genus Rhizobium,as described in Belgian patent specification No. 671,692, or an esterasederived from orange peel, as described in British specification No.966,222.

3-halomethy1-7B-acylaminoceph-3 em 4 carboxylic acids, the halogen atombeing chlorine, bromine, or iodine and. salts thereof e.g. with alkalimetals such as sodium or potassium and with organic bases, andestersthereof, where the esterifying group is preferably one which isreadily introducible and removable without appreciable A Aisomerization, are novel compounds and constitute a feature of theinvention.

OXYGEN LEAVING-GROUPS Another class of starting materials useful in theprocess according to the invention has the partial formula (where Q isas hereinbefore defined). This class includes estersof acetic acidderivatives having at least one elec tron-withdrawing substituent on thea-carbon atom, and estersgof nuclear substituted benzoic acids, thenuclear substituent also being of the electron-withdrawing type. Thusthe acid HX may be any of the various haloacetic acids (di'chloroaceticacid being particularly preferred), methoxy-, alkylthio-, orcyano-acetic acid, glyoxylic acid, phenylpropiolic acid, a hemi-ester ofmalonic or oxalic acid, phenylglyoxylic acid, or a substitutedphenylglyoxyliciacid, the substituent(s) being for example one or morehalogen atoms (F, Cl, Br, or 1), methoxy groups or methylj groups. Whenthe acid HX is a substituted benzoic acid, the choice of the substituentin the benzene ring will be influenced to some extent by a considerationof the stereochemistry of the benzoic acid HX. In general we prefer thephenyl group to be substituted in the 3- or 4- position rather than the2-position since substitution of the 2-position gives rise to themaximum steric hinderance. Thus possible substit-uents include, forexample, 4-methyl, 3-chlor'o or bromo, 3, or 4-nitro or 3,5-dinitro, 3-,or 4-tri-fluoromethyl, 4-carbamoyl, 3-, or 4-(esterified carboxyl), or3-, or 4-cyano.

The acid HX may also be formic acid.

When the group X contains a reactive centre, e.g., an active chlorineatom as in a dichloroacetoxy group, the incoming nucleophile may attackat this centre also. Under such circumstances, it is desirable to use acorresponding excess of the incoming nucleophile, e.-g., two equivalentsexcess when dichloroacetoxy is displaced.

The starting materials having the partial formula (where Q is ashereinbefore defined) are conveniently prepared by acylation of acephalosporanic acid derivative having a S-hydroxymethyl group. Theacylating agent is preferably a reactive derivative of an acid such as aketen (where this exists), an acid halide, e.g. a chloride or bromide,an anhydride or mixed anhydride, e.g., with pivalic acid or formed witha haloformate, or an active ester or azide; alternatively, the aciditself can be used, together with an esterifying agent, e.g.,carbonyldiimidazole or a carbodiimide such as N,N-diethyl-, -dipropyl-,or -diisopropyl-carbodiimide, or preferablyN,N-dicyclohexyl-car'bodiimide.

The acylation should be effected under such conditions that both lactoneformation and A A isomerisation are substantially avoided. Lactoneformation can be reduced by esterification of the 4-carboxy group beforeacylation. The esterifying group should be readily introducible andremovable without resulting in A*- A isomerisation. As mentioned abovepreferred protecting groups are the diphenymethyl group and the5,5,[3-trichloroethyl group which can be readily introduced and removed(after acylation of the 3-CH OH group) substantially without A A2isomerisation.

The esterifying group is preferably removed before the cephalosporanicacid derivative is reacted with the incoming nucleophile.

3 haloacetoxymethyl 75' acylamidoceph-3-em-4-carboxylic acids, thehalogen atom(s) being chlorine bromine or iodine, and3-formyloxymethy1-7,8-acylaminoceph-3-em- 4-carboxylio acids and saltsthereof e.g. with alkali metals such as sodium or potassium and withorganic bases, and esters thereof, where the esterifying group ispreferably one which is readily introducible and removable withoutappreciable A A isomerisation, are novel compounds and constitute afurther feature of the invention.

NITROGEN LEAVING-GROUPS A further class of starting materials useful inthe process according to the invention has the partial formula (where Qis as hereinbefore defined), a preferred group of compounds of this typebeing isothiocyanates having the formula It is desirable that the4-carboxy group should not be esterified but be present as CW or COO'Hwhen X represents an oxygen or nitrogen leaving group.

INCOMI-NG NUCIJEOPHILESJ Nitrogen nucleophiles (a) NR R R in which R Rand R, which may be the same or dif ferent are substituted orunsubstituted aliphatic, araliphatic or aromatic groups; any twotogether with the nitrogen atom if desired forming a heterocyclic ringwhich may be interrupted by one or more further hetero-atoms;

in which n is 0 or an integer from 1 to 5 and R Which when n is from 2to 5, may be the same or different, is an aliphatic, aryl or araliphaticgroup or an alkoxyor acyloxymethyl, formyl, carbamoyl, acyloxy,esterified carboxyl, alkoxy, aryloxy, aralkoxy, alkylthio, arylthio,aralkylthio, cyano, hydroxy, N-monoloweralkyl carbamoyl,N,N-diloweralkyl-carbamoyl, N (hydroxyloweralkyl)carbamoyl, orcarbamoylloweralkyl radical.

in which R is as defined in (b) and m is 0 or an integer from 1 to 4,

N E j in which R and m are as defined in (c),

in which R and mare as defined in (c), (fl

n N a Kw N is in which R is as defined in (b), p is or an integer from 1to 3, and R' is an aliphatic, araliphatic, aryl, or acyl radical or ahydrogen atom.

(a) in P /N in which R R and p are as defined in (f),

d) e (R in which R R and p are as defined in (f),

(R w-Fl? N ie in which R and R are as defined in (f) and q is 0, l or 2,(J') in which R and q are as defined in (i),

in which R and p are as defined in (f), and

in which R and p are as defined in (f).

Carbon nucleophiles Examples of carbon nucleophiles include cyanides,pyrroles and substituted pyrroles, e.g., indoles, and compounds givingstabilised carbanions, for example, acetylenes and compounds havingS-diketone groups, for example, acetoacetic and malonic esters andcyclohexane-1,3- diones.

Thus the term carbon nucleophile includes compounds of the followingformulae:

in which M is a metal cation, preferably an alkali metal or alkalineearth metal cation or a quaternary ammonium ion, and v is the valency ofthe cation.

in which R is an aliphatic, araliphatic or aryl group or an esterifiedcarboxy, acyloxy or acyl group, p is O or an integer from 1 to 3, and Ris an alkyl, aralkyl, or aryl group or a hydrogen atom, at least one ofthe B-positions being unsubstituted,

in which R and R are as defined in (b) and n is 0 or an integer from 1to 5, the 3-position being unsubstituted,

in which the group R, which may be the same or different, are hydrogenatoms or alkyl, aralkyl or aryl groups and R is an alkyl, aralkyl, aryl,alkoxy, aralkoxy or aryloxy group.

where R is an electron donating group or atom and n is 0 or an integerof from 1 to 5.

Sulphur nucleophiles Examples of sulphur nucleophiles include thioureaand aliphatic, aromatic, araliphatic, alicylic and heterocyclicsubstituted thioureas, aromatic and aliphatic thioamides, for examplethioacetamide and thiosemicarbazide, and thiosulphates.

Thus the term sulphur nucleophile includes compounds of the formulae:

in which R R R and R which may be the same or different, representhydrogen atoms, aliphatic, alicyclic, aromatic, araliphatic orheterocyclic groups, or R and R together form a divalent group. R or Rmay alternatively be a group NR R where R and R are as defined above.

in which R is a straight or branched chain aliphatic or araliphaticgroup, and R and R which may be the same or different, are aliphatic,araliphatic, acyl or aryl groups or hydrogen atoms.

in which M is a metal cation, preferably an alkali or alkaline earthmetal cation, or a quaternary ammonium ion, and v is the valency of thecation.

Oxygen nucleophiles Examples of oxygen nucleophiles include water andalcohols, for example alkanols such as methanol, ethanol, propanol andbutanol. Water furnishes both H 0: and OH- and is thus a competitornucleophile in any reaction occurring in aqueous medium.

The term oxygen nucleophile thus includes compounds of the followingformula:

HOR

in which the group R may be lower alkyl (e.g. methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl etc.); lower alkenyl (e.g. vinyl, allyl,isopropenyl, etc.); lower alkynyl (e.g. ethynyl, propynyl, etc.); lowercycloalkyl (e. g. cyclopentyl, cyclohexyl, etc.); lower cycloalkyl loweralkyl (e.g. cyclopentylmethyl, cyclohexylethyl etc.); aryl (e.g. phenylor naphthyl); aryl lower alkyl (e.g. benzyl); heterocyclic; heterocycliclower alkyl (e.g. furfuryl) or any of these groups substituted by, forexample, one or more of lower alkoxy( methoxy, ethoxy, etc.), loweralkylthio (methylthio, ethylthio etc.), halogen (chlorine, bromine,iodine or fluorine), lower alkyl (methyl, ethyl etc.), nitro, hydroxy,acyloxy, carboxy, carbalkoxy, lower alkylcarbonyl, lower alkylsuphinyl,lower alkylsulphonyl, lower alkoxysulphonyl, amino, lower alkylamino oracylamino groups.

We particularly prefer those compounds in which the group R is a loweralkyl group. These 7/3-acylamido-3- loweralkoxymethyl ceph 3em-4-carboxylic acids have shown interesting properties as orallyadministrable antibiotics as is described in copending application ofJames Kennedy et al. Ser. No. 752,191, filed on even date. Compoundsaccording to the invention described in application Ser. No. 752,191which are particularly interesting as antibiotics are those of theformula s Ar-cacouu X i n CH R1 coon where Ar is an aromatic group e.g.phenyl or phenyl substituted with halo, hydroxy, lower alkyl, nitro,amino, lower alkanoyl, lower alkoxy or lower alkylmercapto, or aheterocyclic group, e.g. thien-2-yl or thien-3-yl, X is amino,substituted amino [e.g., acylamido or a group obtained by reacting theamino group and/or acylamido group (s) with an aldehyde or ketone e.g.acetone or methyl ethyl ketone}, hydroxy, formyloxy or loweralkanoyloxy, and R is a lower alkyl group, and non-toxic derivativesthereof. Of particular interest are the compounds of the Formula I wherethe acid ArCH(X)COOH is of the D-series.

REACTION CONDITIONS The reaction may conveniently be effected bymaintaining the reactants in solution at a moderate temperature, e.g.,0-120" 0., preferably 35-75 C., advantageously about 50 C. Reactions areusually complete (in aqueous solvents) in about 15 min. at 50, and incorrespondingly longer times at lower temperatures or correspondinglyshorter times at higher temperatures. The reaction is advantageouslyeifected using from one molar equivalent to ten molar equivalents ofincoming nucleophile. The pH value of the reaction solution underaqueous conditions is advantageously maintained within the limits 5-8,preferably 6-7. If necessary the pH of the solution should be adjustedto the desired value by the addition of a butlering agent such as sodiumacetate. When working under non-aqueous conditions, the reaction mediumshould be neither extremely basic nor extremely acidic.

Since the reaction appears to proceed by a polar or ionic mechanism itis desirable to employ a polar medium for the reaction to proceed. Themost generally suitable is water but organic solvents such as dioxan,ethyl acetate, formamide, N,N-dimethylformamide or acetone may beemployed. The organic solvents may be used in the presence or absence ofwater.

Where essentially non-aqueous conditions are used the alcohol or phenolmay itself be used as the reaction medium and this is advantageous inthat the absence of water from the reaction medium precludes acompetitive reaction between the water and the cephalosporin moleculetaking place. Non-polar solvents may also be used, in which cases theaddition of as little as 0.5% of Water will often bring about thedesired amount of polarity. In certain cases the nucleophile itself maybe the solvent,

particularly when the ethers are being prepared from alcohols.

Organic media which may be used include lower alkanoic acid nitrilese.g. acetonitrile or propionitrile; halogenated hydrocarbons e.g.methylene chloride, carbon tetrachloride, chloroform, ethylenedichloride or perchloroethylene; lower nitroalkanes, e.g. nitromethane;nitro-aromatic compounds, e.g. nitrobenzene; cyclic ethers e.g. dioxanor tetrahydrofuran; amides of the general formula R .CO.NR R' where R isa hydrogen atom or an alkyl group containing 1 to 5 carbon atoms and Rand R", which may be the same or different, are each a hydrogen atom oran alkyl group containing 1 to 5 carbon atoms, or, alternatively R and Rtogether form a divalent aliphatic group which, together with theadjacent nitrogen atom, forms a heterocyclic ring. Examples of amides ofthis type are N,N-dimethylformamide, N,N- diethylformamide,N,N-dimethylacetamide, formamide and N-methylformamide. Other solventswhich may be used include N-lower alkyl pyrrolidones e.g.N-methylpyrrolidone and di-lower alkyl sulphoxides, e.g.dimethylsulphoxide.

The reaction medium need not be liquid at room temperature. Solids, e.g.acetamide, may be used so long as they are liquid at the reactiontemperature.

The reaction product may be separated from the reaction mixture, whichmay contain, for example, unchanged cephalosporin and other substances,by a variety of processes including recrystallization, ionophoresis,paper chromatography or by chromatography on ionexchange resins.

ACYL GROUPS Whilst R may represent an acyl group in general terms, onemay use specific acyl radicals as defined in the following generalformulae, but it should, however, be noted that this is not intended tobe an exhaustive list of all the possible N-acyl groups which may bepresent.

(i) RC I-I ,,CO'where R is aryl (carbocyclic or heterocyclic),cycloalkyl, substituted aryl, substituted cycloalkyl, or a non-aromaticheterocyclic group, and n is an integer from 14. Examples of this groupinclude phenylacetyl, substituted phenylacetyl, e.g.,fluorophenylacetyl, nitrophenylacetyl, acetoxyphenylacetal,alkanoylphenylacetyl, or hydroxyphenylacetyl, thienyl-Z- and -3- acetyl,4-isoxazo1yland substituted 4-isoxazolyl-acetyl and pyridylacetyl.

The substituted 4-isoxazolyl group may be a 3-aryl-5-methyl-isoxazol-4-yl group, the aryl group being e.g. phenyl orhalophenyl, e.g., chloroor bromophenyl. An acyl group of this type is3-o-chlorophenyl-5-methylisoxazol-4acetyl.

(ii) C H CO-where n is an integer from 1-7. The alkyl group may bestraight or branched, and if desired, may be interrupted by an oxygen ora sulphur atom or substituted by e.g. a cyano group. Examples of suchgroups include hexanoyl, heptanoyl, octanoyl, butylthioacetyl, andcyanoacetyl.

(iii) C I-I COwhere n is an integer from 2-7. The alkenyl group may bestraight or branched and, if desired, may be interrupted by an oxygen ora sulphur atom. Examples of such groups include acrylyl, crotonyl andallylthioacetyl.

(iv) RO.CH .COwhere R has the meaning defined under (i). An example ofsuch a group is phenoxyacetyl.

(v) R SCH .COwhere R has the meaning defined above. Examples of suchthio groups include S-phenylthioacetyl, S-chlorophenylthioacetyl andS-bromophenylthioacetyl.

(vi) R(OH ),,Z(CH .CO-where R has the meaning defined above, m is aninteger from 1-5, It is an integer from l-4, and Z is an oxygen orsulphur atom. Examples of such groups include S-benzylthioacetyl,S-benzylthiopropionyl and S-phenethylthioacetyl.

(vii) R"COwhere R has the meaning defined above. Examples of such groupsinclude benzoyl, substituted 9 benzoyl (e.g., aminobenzoyl),4-isoxazolyland substituted 4-isoxazolyl-carbonyl, andcyclopentanecarbonyl. Where the benzoyl group is substituted thesubstituents may for example be alkyl or alkoxy and may be in the 2- or2- and 6-p0sitions; an example of such a group is 2,6- dimethoxybenzoyl.Where the group R represents a substituted 4-isoxazolyl group, thesubstituents may be as set out under (i) above. An acyl group of thistype is 3-o-chlorophenyl-S-methyl-isoxazol-4-yl-carbonyl.

(viii) Amino acyl, for example R CH(NH (CH CO or NH .Ar(CH CO where n isan integer from 1-10, R is a hydrogen atom or an alkyl, aralkyl orcarboxy group or a group as defined under R above, and Ar is an arylenegroup, e.g. p-phenylene or 1,4-naphthylene. Examples of such groups aredisclosed in British patent specification No. 1,054,806. Groups of thistype disclosed by the British patent are p-aminophenylacetyl,waminoacetyl-u-amino on rnethylacetyl, a-amino-a-phenylacetyl,p-aminopropionyl, fi-amino-B-phenylpropionyl, L-y-amino-'y-carboxybutyryl, 11aminoundecylyl and d-l-,8-amino-pcarboxypropionylgroups. Other acyl groups of this type include those, e.g.fi-aminoadipoyl, derived from naturally occurring amino acids.

where the groups R which may be the same or diiferent, are hydrogenatoms or monovalent organic groups, e.g., lower alkyl or halogensubstituted lower alkyl.

'(x) Substituted glyoxylyl groups of the formula R .a-CO.CO'where R isan aliphatic, araliphatic or aromatic group, e.g. an aromatic grouphaving an atomic weight sum greater than 78, e.g., a thienyl group or amono-, di-, or tri-substituted phenyl group, the substituents being, forexample, one or more halogen atoms ('F, Cl, Br or I), methoxy groups,methyl groups or amino groups, or a fused benzene ring. Included in thisgroup are also the u-carbonyl derivatives of the above glyoxylyl andsubstituted glyoxylyl groups, formed for example with hydroxylamine,semicarbazide, thiosemicarbazide, isoniazide or hydrazine.

(xi) a-Substituted carboxylic acid acyl groups, where the m-substituentis an amino, substituted amino [e.g. acylamido or a group obtained byreacting the amino group and/or acylamido group(s) with an aldehyde orketone e.g. acetone or methyl ethyl ketone], hydroxy, carboxy,esterified carboxy, cyano, halogeno, acyloxy (e.g. formyloxy or loweralkanoyloxy) or etherified hydroxy group. The carboxylic acid may bealiphatic, e.g., an a-substituted paraifinic acid, or araliphatic, e.g.,an rat-substituted phenylacetic acid. Acyl groups of this characterinclude the group ArCH (X)CO where Ar and X have the meanings definedabove.

The following exampels illustrate the invention. In the examples:

System A is descending n-propanol-water=7:3, on Whatman No. 1 paper atroom temperature.

System B is butanol:ethanol:water=4:1:5, equilibrated at roomtemperature, the upper phase being used as developer in descendingmanner, in equilibrium With lower phase, on Whatman 3MM paper bufferedto pH 6 with 0.05 M sodium dihydrogen phosphate.

System C is ethyl acetate:butanol:0.1 M-sodium acetate, pH :8:128,equilibrated at 38 C., the upper phase being used as developer indescending manner, in equilibrium with lower phase at 38, on No. 1Whatman paper buffered to pH 5 with 0.1 M sodium acetate.

R represents the R value divided by that of 3-acetoxymethyl713-(2-thienylacetarnido)ceph-3-em-4-carboxylic acid.

The conditions for electrophoresis are those described by Cocker et al.,J. Chem. Soc. 1965, 5015.

Organic solutions were dried over desiccated magnesium sulphate.

Cephaloridine was detected by the blue colour it gave with the potassiumiodoplatinate spray reagent described in Chromatography, E. Merck A.G.,Darmstadt, page 133.

EXAMPLE 1 Preparation of N-[7B-(2-thienylacetamido)-ceph-3-em-4-ylmethyl]-pyridinium-4-carboxylate The title compound wasprepared via the following reaction scheme (the steps are described inmore detail below) 3-hydroXymethyl-7 8=(2-thienylacetamido)-ceph-3-em-4-carboxylic acid Diphenylmethyl3-hydroxymethyl-7fi-(2'-thienylacetamido)-ceph-3-em-4-carboxylateDiphenylmethyl 3-dichloroacetoxymethyl-Hi-(2'-thienyl acetamido-ceph-3-em-4-carboxylate3-dichloroacetoxymethyl-713-(2'-thienylacetamido)-ceph-3-em-4-carboxylic acid N-[7p-(2'-thienylacetamido)-ceph-3-em-3-ylmethyl]-pyridinium-4-carboxy1ate.

(a) Diphenylmethyl 3-hydroxymethyl-7fl-(2 -thienylacetamido) ceph3-em-4-carboxylate.3-hydroxymethyl-7B( 2'-thienylacetamido)eeph-3-em-4carboxylic acid (500 mg.) was dissolved in dry tetrahydrofuran andtreated with a solution of diphenyldiazomethane (300 mg., 1.1 equiv.) inpetrol. Nitrogen was e'volved slowly, and after 2. /2 hours the solutionwas evaporated, the residue dissolved in ethyl acetate, and the solutionwas washed with bicarbonate solution and re-evaporated. This gave a gumwhich solidified on trituration with ether (0.5 g.). A sample wasrecrystallised from methanol, M.P. 164, [a] =+'25 (c, 1.0, dioxan), +22(c, 1.0, tetrahydrofuran). M ethanol 234 nm.

vmax, (bromoform) 3420 (OH), 3280 (NH), 1750 (13- lactam), 1722 cm.-(COOR). (Found (percent): C, 62.2; H, 4.5; N, 5.4; S, 12.1. C H N O Srequires (percent): C, 62.3; H, 4.7; N, 5.4; S, 12.3). R =0.83(Kieselgel G plates; ethyl acetate:benzene=1:2).

(b) Diphenylmethyl 3-dichloroacetoxymethyl-7l3- (2'-thienylacetamido)ceph 3 em-4-carboxylate.--Diphenylmethyl 3hydroxymethyl-7fl-(2-thienylacetamido)ceph- 3-em-4-carboxylate (2.08 g.,4 mmole) and pyridine (1.58 ml.; 20 mmole) were dissolved in drytetrahydrofuran (100 ml.) and cooled to 20. Dichloroacetyl chloride(2.95 g.; 1.96 ml.; 20 mmole) in dry tetrahydrofuran (5 ml.) was addeddropwise. 15 minutes after the addition the mixture was filtered andevaporated, and the residue was partitioned between ethyl acetate andsodium bicarbonate solution. The organic layer was washed with brine,dried and taken to small volume; this solution was then added dropwiseto petrol to give a white solid (2.1 g.; M.P. 60 (softens), [013 17.5(c. 1.14, dioxan), M (ethanol) 236 nm. (e: 13,300, 259 nm. (e=7,600), u(CHBr 1783 (B-lactam), 1760 (CO CHCIZ), 1725 (COOR), 1680 and 1510 cm.-(CONH). N.M.R. spectrum (CDCl )COCHCl 4.131, R =O.37 (Kieselgel G plate;benzenezethyl acetate=5 :1).

(c) 3-dichloroacetoxymethyl-7fl-(2'-thienylacetamido)ceph-3-em-4-carboxylic acid.Diphenylmethyl3-dichloroacetoxymethyl-7fl-(2 thienylacetamido)ceph-3-em-4- carboxylate(6.8 g.) was dissolved in anisole (5 ml.),

11 and trifiuoroacetic acid (15 ml.) was added. After 4 minutes thesolvent was removed at 30. The residue was dissolved in ethyl acetateand re-evaporated; the gum was redissolved in ethyl acetate (10 ml.) andadded dropwise, with stirring, to petroleum ether (400 ml.). The productwas obtained as a yellow solid, M.P. 60 (softens; decomp. at 99) (4.92g.; 95%), [u] =+56' (c., 0.7; dioxau), M (ethanol) 237 nm. (e=12,500),259 nm. (e=7,500), v (CI-IBr 3390 (NH), 1788 (B-lactam), 1760 (000R),1685 and 1518 (CONH), 1735 and 1715 cmr (COOH). N.M.R. spectrum (CDCl)-O.COCHCl 4.07.

The dicyclohexylamine salt crystallised from acetone, M.P. 100 (softens;decomp. at 210). [u] =+36 (c. 1.0, chloroform), M (ethanol) 235 nm.(e=13,600), 265 nm. (e=6,950), u (CI-IBr 1774 (fl-lactam), 1765 (COOR),1635 (000*), 1680 and 1518 (CONH), 812 CIIL'I (CHCI N.M.R. spectrum(CDCl )-CH .O.COCHCl 3.91-r. (Found (percent): C, H, N, C1, 14-2. C H ClN O S (C H NH requires (percent): C, 52.0; H, 5.8; N, 6.5; 01, 14.8.)

(d) Reaction of3-dichloroacetoxymethyl-Ifl-(2'-thienylacetamido)ceph-3-em-4-carboxylicacid with pyridine-This reaction was shown to proceed in dioxan, ethylacetate, methanol, dimethylformamide, formamide and water to givecephaloridine. With the assay procedure described by Taylor (J. Chem.Soc., 1965, 7020), the reaction appeared to give the highest yields inthose sol vents of high dielectric constant.

Dielectric Maximum constant yield Solvent at 25 0. (percent) Dioxan 2. 2l

Ethyl acetate 6. 0 3 Methanol 32. 6 21 N,N-dlmethyllormamide 36. 7 20Formamide 100 53 Water 78. 44

The reaction was shown to proceed at temperatures between 25 and 75". Itwas also shown that the reaction proceeded with increased efliciency inthe non-polar solvents as the percentage of water in these solvents wasincreased.

The reaction was most efiicient when 2 equivalents of pyridine wereemployed.

EXAMPLE 2 Preparation of N-[7;8-(2'-thienylacetamido) ceph-3-em-4-ylmethyl]-pyridinium-4-carboxylate The title compound was preparedvia the following reaction scheme (the steps are described in moredetail below) Diphcnylmethyl 3-hydroxymethyl-7fl-(Z-thienylacetamido)-ceph-3-em-4-carboxylate Diphenylmethyl3-formyloxymethyl-7B-(2'-thienylacetamido)-ceph-3-em-4carboxylate Jr (b)3-formyloxymethyl-7fi- (2'-thienylacetamido)-ceph- 3-em-4carboxylic acidOn reaching room temperature, the mixture was filtered, concentrated,and partitioned between ethyl acetate and 2 N-hydrochloric acid. Onwashing with more acid the ethyl acetate solution precipitated someproduct (1.77 g.); the remaining ethyl acetate was dried and evaporatedto give more product (1.32 g.). A sample of the product wasrecrystallised from ethyl acetate, M.P. 189, u (CHBr 1792 (fi-lactam)1730 and 1232 (COOR), 1690 and 1518 cum- (CONH). N.M.R, spectrum(dimethylsulphoxide) CHO at 1.791. (Found (percent): C, 61.7; H, 4.4; N,4.8. C H N O S requires (percent): C, 61.3; H, 4.4; N, 5.1.) R =0.7(Kieselgel G plate, benzene ethyl acetate-:2: 1.)

(b) 3-formyloxymethyl-7B-(2'-thienylacetamido)ceph- 3-em4-carboxylicacid.-Diphenyhnethyl 3-formyloxymethylJB-(2-thienylacetamido)ceph3-em-4-carboxylate (1.5 g.) was suspended in anisole (5 rnl.), andtri'fluoroacetic acid (10 ml.) was added. After 4 minutes the mixturewas evaporated to dryness at 0.1 mm. The residual solid was washed withether to give the title compound (960 mg., 91%). A sample wasrecrystallised from methanol-petrol, M.P. (softens; decomp. at 107), M(pH 6phosphate buffer) 233 nm. (e=13,200), 260 nm. (e=6,800). Found(percent)): C, 46.5; H, 3.9; N, S, C15H14N2O6S2.1/2H2O requires(percent): C, 46.1; H, 3.8; N, 6.9; C, 16.3.

Sodium salt: M (pH 6-phosphate buffer) 232 nm. (e=13,000), 2579 nm.(e=-6,300), v (Nujol) 2 (fl-lactam), 1718 and 1170 (formate), 1608(000), 1660 and 1538 cm? (OONH). N.M.R. spectrum (D 0), OHO at 1.81; R=0.7 (System 0). Found (percent): C, 46.5; H, 3.9; N, 6.5; S, 16.02. c-H N ogs x /zH O requires (percent): C, 46.1; H, 3.8; N, 6.9; S. 16.3.

(c) N [7B (2' thienylacetamido)ceph-3-em-3-ylmethyljpyridinium-4-carboxylate. 3-formyloxymethyl- 7 8(2' thienylyacetamido)-ceph 3-em-4-carboxylic acid (38 mg.) was kept at38 in water (1 ml.) containing pyridine (0.08 mL). After 1, 2, and 3hours, aliquots (5 a1.)- were subjected to electrophoresis at pH 1.9 andto paper chromatography in system C. The formation of cephaloridine wasestablished by comparison with a standard spot of cephaloricline on theelectrophoretograms and chromatograms, and by spraying the papers withiodoplatinate reagent, which gives a blue colour with cephaloridine.

EXAMPLE 3 Reaction of 3-acetoxy-, 3-chloroacetoxy-, 3-dichloroacetoxy-,3-trichloroacetoxyand 3-phenylglyoxylyloxymethyl 7B(2'-thienylacetamido) ceph-3-em-4-carboxylic acids with (a) aqueous baseand (b) aqueous pyridine The 3 chloracetoxymethyl, 3trichloroacetoxymethyl and 3-phenylglyoxylyloxymethylcompounds wereprepared by adding pyridine (4 equivalents), or a solution of pyridine(4 equivalents) in dry peroxide-free tetrahydrofuran dropwise withstirring at room temperature to a solution of diphenylmethyl 3hydroxymethyl-7B-(2'- thienylacetamido)ceph-3-em-4-carboxylate and theappropriate acid chloride (4 equivalents) in dry peroxide-freetetrahydrofuran. The mixture was stirred for 30 minutes; theprecipitated pyridine hydrochloride was removed and the filtrateevaporated to dryness. The residue was dissolved in ethyl acetate andthe solution washed with saturated sodium bicarbonate solution to removeunreacted acid chloride. Further washing with dilute hydrochloric acidand brine was carried out before the solution was dried (MgSO andevaporated. If the residue could not be induced to crystallise, it wasdissolved in ethyl acetate and the product was obtained in solid form byslowly adding this solution to a large volume of petroleum ether.

In a manner analogous to that described in Example 1(0) thediphenylmethyl esterifying group was removed by acid hydrolysis usingtrifluoroacetic acid in conjunction with anisole.

(a) 3 acetoxymethyl-7fi-(2-thienylacetamido)ceph-3- em -4 carboxylicacid was dissolved in aqueous 0.5 N-sodium bicarbonate; it remainedunchanged. However, the remaining compounds in the title wereinstantaneously converted under the same conditions into products withthe same R values as3-hydroxymethyl-7fi-(2-thienylacetamido)ceph-3-em-4-carboxylic acid.

(b) The compounds described in the title (38 mg.) were each kept at 38in water (1 ml.) containing pyridine (0.08 ml.). After 1, 2, and 3hours, aliquots ,ul.) were subjected to electrophoresis at pH 1.9 and topaper chromatography in system C. The formation of cephaloridine wasestablished by comparison with a standard spot of cephaloridine on theelectrophoretograms and chromatograms, and by spraying the paper withiodoplatinate reagent, which gives a blue colour with cephaloridine.

After 3 hours therew as only a trace of cephaloridine produced from 3acetoxymethyl 7;? (2 thienylacetamido)ceph-3-em-4-carboxylic acid, butwith all of the other compounds conversion to cephaloridine was completein 1 hour and appeared to be efficient.

EXAMPLE 4 Preparation of cephaloridine 3 dichloroacetoxymethyl 7B(2'-thienylacetamido)- ceph-3-em-4-carboxylic acid (1.0 g., 2.15 mmole)was suspended in water 4 ml.), and pyridine (0.352 ml., 4.3 mmole) wasadded. The solution was heated at 50 for 1 hour, acidified to pH 4.5with acetic acid, and washed with ethyl acetate and finally with petrol.The aqueous solution was freed from dissolved organic solvents andapplied to a column comprising (from top to bottom) layers ofdeactivated neutral alumina (8 m1.), Dowex 1 (AcO-) ml.) and Zeokarb 226(H+) (5 ml.). Elution with water was carried out until the eluate showedno optical rotation; the total eluate was freeze-dried and the residuetriturated with methanol to give a white solid (296 mg), [111 +488 (c.0.8, water), M (water) 238 nm.

The product had an infrared and N.M.R. spectrum identical with authenticcephaloridine. It also had identical chromatographic and electrophoreticbehaviour.

EXAMPLE 5 (a) 3-isothiocyanatomethyl-7/8-(2'-thienylacetamido)-ceph-3-em-4-carboxylic acid 3 dichloroacetoxymethyl 7B(2-thienylacetamido)- ceph-3-em-4-carboxylic acid (2 g.) was dissolvedin acetone (30 ml.) containing water (0.3 ml.), and potassiumthiocyanate (2 g.) was added. The mixture was kept at 47 for 30 minutesand then poured into 2 N-hydrochloric acid (20 ml.). The product wasextracted into ethyl acetate; washing with saturated sodium bicarbonatesolution transferred the product to the aqueous layer, from which it wasisolated by acidification and extraction into ethyl acetate. The organiclayer was dried and concentrated and the solution then poured intostirred petrol; the product separated as a pale yellow solid (600 mg.).Of this solid, 500 mg. was dissolved in acetone and treated with sodium2-ethyl-hexanoate solution (1.1 equiv.); on addition of ether, thesodium salt of the title compound precipitated. This salt was dissolvedin water and filtered through neutral alumina, and the eluate wasfreeze-dried to give a white solid (400 mg), [a] =+29 (c. 1.0, water), M(pH 6 phosphate buffer) 236 nm. (6: 14,200), 260 nm. (e=8,600), v (ofthe acid) (C-HBr 2090 (N=C4), 1790 (B-lactam), 1730 (COOH), 1682 and1518 cm. (CONH). N.M.R. spectrum (D 0) CH N=C:S

at 5.501. Found (percent): C, 39.3; H, 3.3; N, 8.4. C H N O S Na2l 2H Orequires (percent): C, 39.0; H, 3.7; N, 9.0. R =2.05 (system C), 1.35(system B). The product reacted in dimethylformamide with sodium azide,

14 evolving a gas, so probably confirming the presence of anisothiocyanate group. (Lieber, Chem. and 1nd, 1958, 1234).

(b) Reaction of 3 isothiocyanatomethyl-7B-(2-thienylacetamido)ceph-3-em-4-carboxylic acid with pyridine The isothiocyanate (5 mg.) in water(0.4 ml.) containing pyridine (0.08 ml.) was kept at 50 for 1 hour. Analiquot of the reaction mixture was subjected to electrophoresis at pH1.9 and to paper chromatography and shown to contain a component whichhad the same migration and chromatographic behaviour as cephaloridine.

EXAMPLE 6N-[7,6-(2'-thienylacetamido)ceph-3-em-3-ylmethyl1triethylammonium-4-carboxylate3 dichloroacetoxymethyl 7B (2' thienylacetamido) ceph-3-em-4-carboxylicacid (1 g.) was dissolved in wateracetone (05:99.5) (10 ml), andtriethylamine (0.55 ml.: 2 equivs) was added. The mixture was kept atroom temperature for 30 minutes and then poured into ethyl acetate;water and acetic acid were added simultaneously so that the pH did notexceed 4.0. The aqueous solution, after washing with ethyl acetate, waspassed through a Dowex 1 (AcO) column and the eluate freeze-dried. Thelyophilised solid was dissolved in methanol and precipitated by pouringthis solution into ether. This gave a white solid mg.), [oc] =+93 (c.1.15, water), )tk (water 237 nm. (e=l2,600), 260 nm. (5:9,000) N.M.R.spectrum systems centered at 6.73 and 8.721: (Found (percent): C, H, N,c2oH27N304S23H20 requires (percent): C, 48.9; H, 6.7; N, 8.6.) Onelectrophoresis at pH 7.0 to the compound showed no net charge, and atpH 1.9 it moved less than cephaloridine. The compound stained blue withiodoplatinate reagent.

EXAMPLE 7 3-ethoxymethyl-7fl-(2'-thienylacetamido)ceph-3-em-4-carboxylic acid 3 dichloroacetoxymethyl 75- (2' thienylacetamido)ceph-3-em-4carboxylic acid (4.74 g.) was refluxed in ethanol ml.) for 60minutes; the mixture was filtered and the filtrate evaporated to give anoil. Trituration with ethyl acetate gave a solid (354 mg.), which wasdiscarded. The ethyl acetate solution was extracted with sodiumbicarbonate solution; the aqueous layer was then acidified and extractedwith ethyl acetate. On drying and evaporation this solution gave a gum(3.34 g.), which was redissolved in hot ethyl acetate. Addition of afourfold volume of ether gave a solid, which was discarded. The filtratewas concentrated and set aside at 0; a white solid (548 mg.)crystallised out, M. P. 160l65 :(decomp), [a] 157 (c. 0.7,tetrahydrofuran) M (Nal-IOO solution) 237 nm. (e=12,800), 260 nm.(e=8,000), 11 (CHBr 1770 (fi-lactam), 1720 (COOH), 1659 and 1678 cm.-(CONH). N.M.R. spectrum (D 0, with sodium bicarbonate) OCH .CH systemscentered at 6.5 and 8.837. (Found (percent): C, 50.6; H, 4.8; N, 7.1; S,16.3. C H N O' S requires (percent): C, 50.2; H, 4.7; N, 7.4; S, 16.8.)R;=1.5 (System C), 1.35 (System B).

EXAMPLE 8 Preparation of 3-ethoxymethyl-7/3-(2'-thienylacetamido)ceph-3-em-4-carboxylic acid (a) Diphenylmethyl 3 chloromethyl 7/3 (2'thienylacetamido)ceph 3 em 4 carboxylate.-Diphenylmethyl 3 hydroxymethyl7B (2 thienylacetamido) ceph-3-em-4-carboxylate (5.2 g., 10 mmole) andpyridine (4 ml., 40 mmole) in dry tetrahydrofuran (75 ml.) were addeddropwise at 20 to a solution of thionyl chloride (2.38 g., 1.45 ml.; '20mmole) in dry tetrahydrofuran (25 ml.) during one hour. After 15 minutesthe mixture was poured into brine and the product extracted into ethylacetate; the organic extract was dried and concentrated. The concentratewas added dropwise to petroleum ether (B.P. 4060) and the solid product(3.9 g., 73%) was collected. A sample was recrystallised from. ethanol,M.P. 12S-133 (decomp.) [a] =6.5 (c., 1.0, tetrahydrofuran); M (ethanol)235 nm. (e=l3,200), 266 nm. (e=8,000); v (bromoforrn) 3390 (NH), 1785(fl-lactam), 1725 (COOR), 1682 and 1510 cm. (CONH); -r (CDCl 5.63,3-methylene group singlet. (Found (percent): C, 60.7; H, 4.7; N, 4.7; S,11.7; C1, 6.2; C H N O S Cl requires (percent): C, 60.2; H, 4.3; N, 5.2;S, 11.9; C1, 6.6.). Rf -47 (silica plates, benzene :ethyl acetate= l Theabove experiment was repeated using n-butyl chlorosulphite ordimethylaminochlorosulphite instead of thionyl chloride, the pyridinebeing added dropwise to the other reagents at 40 C. After completeaddition of the pyridine, the mixture was allowed to warm to roomtemperature, poured into saturated brine, and worked up as above.

.(b) 3 ethoxymethyl 7 B (2 thienylactamido) ceph 3 em 4 carboxylicacid.Diphenylmethyl 3- chloromethyl 719 (2' thienylacetamido)ceph-3-em-4carboxylate (10.3 g.) was dissolved in ethanol (250- m1.) and kept at 60for 4 /2 hours. The mixture was evaporated to give a froth (11.6 g.),which was dissolved in anisole ml.) and treated with trifluoroaceticacid (70 ml.) for 6 minutes. The solvent was removed at 40/1 mm. Theresidual gum was triturated with ethyl acetate; the solid so obtainedwas discarded. The ethyl acetate solution was extracted with sodiumbicarbonate solution, and the separated aqueous layer was acidified topH 2.5 with phosphoric acid, and re-extracted with ethyl acetate. Dryingand evaporation gave a gum which was taken up in the minimum amount ofethyl acetate, and a fourfold volume of ether was added. The precipitatewas discarded and the filtrate evaporated to give a guru (2.0 g.) whichwas recrystallised from ethyl acetate to give the title compound (140mg) The mother liquor was concentrated and cooled to give a secondimpure crop of product (729 mg). The product had the same R; values asthe title compound.

EXAMPLE 9 3-n-propoxymethyl-7,6-(2'-thienylacetamido)-ceph-3-em-4-carboxylic acid '3 diehloroacetoxymethyl 7 3 (2thienylacetamido)ceph-3-em-4-carboxylic acid (5 g., 12.9 mmoles) wasrefluxed in n-propanol (50 ml.) for 20 minutes. The solution was cooledand the precipitated brown solid was filtered oif and discarded. Thefiltrate was treated with water (300 m1.) and the pH adjusted to 8.5with aqueous sodium bicarbonate. The solution was extracted with ethylacetate (2X 20 m1.) and the extracts were discarded. The aqueous phasewas acidified to pH 1.5 and extracted with ethyl acetate (3X 20 ml.).After drying, the solvent was removed and the residue dissolved in ethylacetate (50 ml.), and ether (100 ml.) was added. 'The precipitated brownsolid was filtered 01? and discarded, and the filtrate evaporated todryness. The residue was twice recrystallised from aqueous ethanol togive the title compound as colourless needles (500 mg, 11.7%), M.P.152154 (decomp.) [a] =79.8 (c. 1.0, tetrahydrofuran), k (pH 6-phosphatebufier) 236 nm. (e=13,600), 260 nm. (e=8,700) (shoulder), v (Nujol) 3310(-NH), 1772 (fl-lactom), 1724 (COOH), 1665 and 1535 cmr (--CONI-I).(Found (percent): C, 51.3; H, 5.1; N, 6.9; S, 15.9. C I-I N S Qr,requires (percent): C, 51.5; H, 5.1; N, 7.1; S, 16.2.) N.M.R. (DO--NaHCO 5.78, 6.60, 8.52, 9.131- (CH OCH CH CH R: 0.48 (System B); 0.54(System C).

16 EXAMPLE 10 The reaction between 3 dichloroacetoxymethyl-7,8-(2-thienylacetamido)ceph-3-em-4-carboxylic acid and various alcohols R: R1System B System 0 Alcohol CHaOH 0. 26 0. 29

CHaCHzOHzC HzOH 0. 46 0. 50

EXAMPLE 11 3 isopropoxymethyl-7fl-(2'-thienylacetamido)ceph-3-em-4-carboxylic acid 3-dichloroacetoxymethy1 7e (2'-thienylacetamido)-ceph-3-em-4-carboxy1ic acid (12.0 g., 25.8 mmole) was refluxed inisopropanol (100 ml.) for 45 minutes. After filtration through a pad ofkieselguhr, the isopropanol solution was poured into water (1000 ml.)and the 'pH adjusted to 8.5. The solution was extracted with ethylacetate (2X 100 ml.) and the extracts discarded. The solution was thenacidified to pH 1.5 with 2 N-hydrochloric acid and extracted with ethylacetate (3x100 ml.). The ethyl acetate extracts were dried overmagnesium sulphate and evaporated. The residue (8.5 g.) was dissolved ina minimum of hot ethanol, from which the title compound separated oncooling (1.5 g., 15%). This material recrystallised from ethanol ascolourless prisms, M.P. 169-171 (d), [a] +87 (c. 1.0, tetrahydrofuran),M (ethanol) 237 nm. (e 14,500), 260 nm. (e 7,900), v (Nujol) 1775(fi-lactam), 1728 (COOH), 1668 and 1535 cm.- (CONH), N.M.R. (D 0, withsodium bicarbonate) 5.64 and 5.95 (quartet: J=16 HZ., --CH OCH(CH 6.31,(8.871) OH --OCH(CH respectively). (Found (percent): C, 51.6; H, 5.1; N,6.7; S, 16.3. C H N O S requires (percent): C, 51.5; H, 5.1; N, 6.1; S,16.2) R; 0.32 (System B), 0.65 (System C).

EXAMPLE 12 The reaction of 3-dichloroacetoxymethy1-7fi-(2'-thieny1-acetamido)-ceph-3-em-4-carboxylic acid with various a1- cohols(estimated the area of biologlcally active zone) the area ofUV.-absorbent zone of the new spots (other than material remaining onthe base line or travelling at the solvent front) are summarised in thetable following Example 13.

EXAMPLE 13 The reaction between3-dichloroacetoxymethyl-7fl-(2'-thienylacetamido)-ceph-3-em-4-carboxylicacid and phe- 1101 A mixture of the acid (0.5 g., 1.08 mmole) and phenol(0.8 g., 5.3 mmole) was heated at for 8 minutes. The melt was cooled anddissolved in ethyl acetate (10 ml.). The insoluble material was filteredoff and discarded, and the filtrate washed with water (2x10 ml.).Samples (5 17 ,ul.) were spotted onto papers which were developed withsolvent System C to about 30 cms. The papers were submitted forbioautograph against Staph. aureus C 864 and E. coli 573. The resultswere estimated in the same manner as in Example 12 and are presented inthe following table.

EXAMPLE 14 N-[7/3-(2 thienylacetamido)ceph-3-em thiazolium-4-carboxylate 3-dich1oroacetoxymethyl 7,6 (2'-thienylacetamido)ceph-3-em-4-carboxylie acid (3 g., 6.5 mmole) was dissolved in50%-aqueous acetone (50 m1.), and treated with thiazole (1.8 ml., 25.4mmole), and the solution heated at 50 for 30 minutes. The acetone wasremoved under vacuum and the aqueous layer extracted with ethyl acetate(2 10 ml.) The dissolved ethyl acetate was removed under reducedpressure and the aqueous residue passed down a column of Dowex 1 (AcO-)(3X 10 ems.) and eluted with water until the eluate did not showsignificant optical rotation. The eluates were freeze-dried. Thefreeze-dried solid was treated with methanol, when it crystallisedwithout going into solution (800 mg., 29% M.P. 169172 (deeomp.) [a]=14.1 (c. 1.03, acetone: water =1.1), A (pH 6 phosphate buifer) 2367 nm.(e=18,000), 264 nm. (e=10,000) (shoulder), v (Nujol) 1760 (fi-lactam),1692 (CONH-), 1602 cm. (COO-). (Found (percent): C, 4-5.6; H, 3.7; N,9.4; S, 21.1. C H N S O J /zH O requires (percent): C, 45.5; H, 4.0; N,9.4; S, 21.4.) R; 0.14 (System B), 0.04 (Sys tem C).

- 3 ylmethyl] EXAMPLE 15 3 (N-pyrazolylrnethy1) -7,8-2-thienylacetamido) ceph-3-em-4-carboxylic acid 4-dichloroacetoxymethyl718 (2 thienylacetamido) ceph-3-em-4-carboxylic acid (3.2 g., 6.9 mmole)was dissolved in 50%-aqueous acetone (60 ml.) and treated with pyrazole(1.6 g., 24 mmole), and the solution heated at 50 for 35 minutes. Theacetone was removed under reduced pressure, and the aqueous residue wasextracted with ether (9x 20 ml.). After drying, the ether was evaporatedand the solid residue recrystallised from aqueous alcohol (1.3 g., 47%),M.P. 180185 (decomp) [a] =53.5 (c. 1.01, ethanol), A (ethanol) 236 nm.(e=14,0), 261 nm. -(e=8,250) (shoulder), v (Nujol) 3272 (-NH), 1773(B-lactarn), 1712 (COOH,( 1660' and 1532 cm? C-ONH). (Found (percent):C, 50.9; H, 4.2; N, 13.9; S, 15.7. C H N S O requires (percent): C,50.5; H, 4.0; N, 13.9; S, 15.9.) N.M.R. (D 0, with NaHCO 2.30, 3.62,2.31-r.

NCH=CHC H-N Rf 0.37 (System B), 0.12 (System C).

EXAMPLE 16 N- [7,8- 2'-thienylacetamido ceph-3 -em-3-ylmethyl] -5(fl-hydroxyethyl)-4"-methylthiazolium-4-carboxylate3-dichloroacetoxymethyl 7B (2' thienylacetamido') ceph-3-em-4-carboxylicacid (1.0 g., 2.15 mmole) and 5- 18 (fl-hydroxyethyl)-4-rnethylthiazole(0.7 g., 4.9 mmole) were dissolved in 50%-a-queous acetone (20 ml.) andthe solution was heated at 50 for 30 minutes. The solution was cooledand the acetone removed under reduced pressure. The aqueous layer wasextracted with ethyl acetate (2X 10 mls.) and passed down a column ofDowex 1 (AcO) (2X 8 cm.), and eluted with water until the eluate did notshow any significant optical rotation. The eluate was freeze-dried togive a white solid (400 mg, 39%), M.P. 125-130 (decomp.) [a] =l4.8 (c.1.01, acetone-water=1:1), A (pH 6phosphate bufier) 305 and 257 nm. (614,700 and 12,000 resp.), v (Nujol) 1775 ([i-lactam), 1670 and 1550(CONH), 1615 (COO-), cm.- (Found (percent): 0, 47.7; H, 4.6; N, 8.1; S,C2 H21N3S305Jfi/2H2O requires (percent): C, 47.7; H, 4.8; N, 8.3; S,19.0.) R 0.16 (System B), 0.04 (System C).

EXAMPLE 17 N-[4-diphenylmethoxycarbonyl-7fi (2-thienylacetamido)ceph-S-em-3-ylmethyl]pyridinium chloride Diphenylmethyl 3-chloromethyl76 (2 trienylacetamido)ceph-3-em-4-carboxylate (1 g.) was kept at 50 indry pyridine (10 ml.) for 2 hours. The pyridine was removed at 30/0.1mm., and the residue partitioned between water and ethyl acetate. Theaqueous layer was freed from organic solvents and freeze-dried to give awhite solid, which was triturated with ether to give the title compound(760 mg., 65%), [a] =+280 (c. 1.0, water), A (pH 6-phosphate buifer) 260nm. (e=14,800), 249-250 nm. (e=13,700) (both inflexions) v (Nujol) 1780(fi-lactam), 1742 (COOR), 1545 and 1680 cm? (CONH). N.M.R. spectrum(pyridine) 3-1- CH at 5.751. (Found (percent): 59.4; H, 4.7; N, 6.1; S,9.9; Cl, 5.4. C H gN S O CL1 /2H O requires (percent): C, 59.5; H, 4.8;N, 6.5; S, 9.9; Cl, 5.5.) The product carries a positive charge at pH1.9 and at pH 7 (electrophoresis). It stains violet with theiodoplatinate spray reagent.

In a manner analogous to that described in Example 1(c) the diphenylmethyl esterifying group was removed by acid hydrolysis usingtrifiuoroacetic acid in conjunction with anisole.

EXAMPLE 18 Diphenylmethyl3-n-propoxymethyl-7B-2'-thienylacetamide-ceph-3-em-4-carboxylate Asolution of diphenylmethyl 7B-(2-thienylacetamido)- 3chloromethylceph-3-ern 4 carboxylate (1.5 g., 2.8 mmole) in acetone (80ml.) was treated with a solution of sodium iodide (0.45 g., 3.0 mmole)in acetone (15 ml.). The solution was allowed to stand in the dark for 1hour at room temperature. The solution was filtered and poured intowater. The mixture was extracted with ether (3x 15 ml.) and the etherextracts dried over magnesium sulphate and evaporated. The gum wasdissolved in n-propanol (50 ml.) and treated with mercuric perchloratehydrate (4 g., 9.6 mmole). After 5 minutes the black precipitate wasfiltered on kieselguhr. The filtrate was evaporated under reducedpressure and the residue was dissolved in ethyl acetate, filtered,washed with aqueous sodium bicarbonate and water, dried, and evaporated.The resulting gum was chromatographed on a silicic acid column (4.5 X 25cms.) in benzene2ethyl acetate=9.1. The fractions containing then-propoxymethyl compound (R ca. 0.7, ethyl aeetate:benzene=1:5, onsilica gel G) were bulked and evaporated (0.6 g., yield ca. 38%).

EXAMPLE 19 Preparation of3-methoxymethyl-7B-(2'-thienylacetamido)ceph-3-em-4-carboxylic acid Thetitle compound was prepared via the following reaction scheme (the stepsare described in more detail below).

1 9 Diphenylmethyl 3-chloromethyl-7 3-(2'-trienylacetamido)ccph-3-em-4-carboxylate iDiphenylmethyl 3 -iodomethyl7;3-( 2 '-tl1ienylacetamido) ceph-3-em-4-carboxylate Diphenylmethyl3-methoxymethyl-7fi-(2-thienylacetamido)ceph-3-em-4-carboxylate3-methoxymethyl-7fl- (2-thienylacetamido) ceph- 3-em-4-carboxylic acid.

(a) Diphenylmethyl 3 iodomethyl-7fi-(2'-thienylacetamido)ceph 3em-4-carboxylate.Diphenylmethyl 3- chloromethyl7B-(2'-thienylacetamide)ceph-3-em-4-carboxylate (7.858 g., 14.5 mmole)was dissolved in acetone and reacted with sodium iodide (7.8 g., 50mmole) in acetone (100 ml.) in the dark for 90 minutes. At the end ofthis time the solution was filtered and poured into water (750 ml.)containing sodium chloride and sodium thiosulphate. The oil wasextracted with ether (4X and the organic layer washed once with waterand twice with brine, and dried and evaporated to a foam, which wascrystallised from ethyl acetate (16 ml.) to give the title compound. Themother liquors were washed with sodium thiosulphate, water and brine,and were dried and evaporated to a foam (2.75 g., impure title compound,approx. 30% yield). Yield of crystalline material 60%, M.P. 155- 161 C.(decomp.), [M 86.3 (c. 0.73; tetrahydrofuran), A (ethanol) 290 nm. (68,400), v (Nujol) 1773 (fi-lactam), 1717 R), and 1670 and 1520 cm.-(CONH-), N.M.R. (deuterochloroform) 1- 5.70 (2- proton broad singlet)(CH I). (Found (percent) C. 51.6; H. 3.7; I, 19.7; N, 4.3; S, 10.2. C HIN O S requires (percent): C, 51.3; H, 3.7; I, 20.1; N, 4.4; S, 10.2.) R0.65 (Kieselgel G, benzene-ethylacetate= :1) (T.L.C.).

(b) Diphenylmethyl 3methoxymethyl-7fl-(2'-thienylacetamido)ceph-3-em-4-carboxylate.-Asolution of diphenylmethyl 3-iodomethyl-718:(2'-thienylacetamide)ceph-3-em-4-carboxylate 2.5 g., 3.96 mmole) in benzene (100 ml.) was treatedwith a solution of mercuric perchlorate (0.9 g. n 4 mmole) in methanol(25 ml.). After standing five minutes at room temperature, the solutionwas successively washed with aqueous sodium hydrogen carbonate, aqueoussodium thiosulphate and water, and then evaporated under reducedpressure. The gum was dissolved in benzene/ethyl acetate (9: 1) and thesolution filtered under reduced pressure through a pad of silicic acid.The pad was then washed with the same solvent. The combined filtrateswere evaporated and the gum dissolved in ethanol ml.). When the titlecompound separated as colourless prisms (0.61 g., 29%), M.P. 146-157",+15.6 ,(c. 1, tetrahydrofuran), A (ethanol) 235 (sh.), 260 (sh.) nm. (614,800, 9,100 respectively), 1 m (Nujol) 1786 p-lactam, 1725 (COOR) 1668and 1646 (CONH) cum- N.M.R. 5.76 CH OCH 6.80 r (-CH OCH (Found(percent): C, 63.1; H, 4.9; N, 5.1; S, 11.5. C H N O S requires,(percent): C, 62.9; H, 4.9; N, 5.2; S, 12.0.)

(c)3 methoxymethyl 7,3-(2'-thienylacetamido)ceph- 3 em 4-carboxylieacid.Diphenylmethyl 3-methoxymethyl7B-(2'-thienylacetamido)ceph-3-em-4-carboxylate (2 g.,) was dissolved ina mixture of trifluoroacetic acid (8 ml.) and anisole (2 ml. and after 5minutes the reagents removed under reduced pressure. The resulting gumwas dissolved in ethyl acetate and poured into 60-80 petroleum and theproduct recovered by filtration. This material crystallised from ethylacetate as colourless prisms, M.P. l53l57 (decomp.), [11],; +816 (c. l,tetrahydrofuran), A (ethanol) 237, 260 (sh.) n. (6 13,700

20 and 7,700 respectively), vmax, ,(Nujol) 1780 (fi-lactam), 1720(COOI-I), 1690 and 1524 cm." (CONH), N.M.R. 5.8-0 (CH OcHa), 6.701- (CHOCH (Found (percent): C, 49.2; H, 4.4; N, 7.5; S, 17.0. C H N O Srequires (percent): C, 48.9; H, 4.4; N, 7.6; S, 17.4.)

EXAMPLE 20 (a) Diphenylmethyl 3- (2'-oxocyclohexyl) methyl-7p-(2"-thienylacetamido)seph-3-em-4-carboxylate A solution ofdiphenylmethyl 3 iodomethyl-7B-(2- thienylacetamido)ceph-3-em-4-carboxylate ,(8 g., 12.7 mmole) in dry benzene was treatedwith N--cyclohex-1- enyl)-pyrrolidine (4 g., 26.2 mmole) and refluxedfor 5 minutes. The solution was then cooled and a mixture of 2N-hydrochloric acid (20 ml.) and acetone ml.) added. The suspension wasWell shaken until all the oil had dissolved and then ethyl acetate (300ml.) and water ml.) added. After shaking, the aqueous layer wasseparated and discarded and the organic layer 'washed with (a) aqueoussodium thiosulphate, ,(b) aqueous sodium bicarbonate, and (c) water, andthen dried and evaporated. The foam was dissolved in ethyl acetate (25ml.) and after 15 minutes the title compound filtered off, washed withethanol and dried (4.5 g., 59%). This material crystallised from ethanolas colourless prisms, M.P. 167-170", [111 +8.5 (c. 1, tctrahydrofuran),M (ethanol) 260 nm. (a 7,500), 11 (CHBr 1780 (B- lactam), 1720 (COOR),1702 =O), and 1682 and 1512 (CONH) cm.- N.M.R. (CDCI) 7.3-9.0 'r(protons in cyclohexanone ring). (Found (percent): C, 65.4; H, 5.4; N,4.5; S, C H N O S requires (percent): C, 65.6; H, 5.4; N, 4.6; S, 10.6.)

(b) Diphenylmethyl 7B-amino-3(2'-oxocyclohexyl)methylceph-3-em-4-carboxylate, hydrogenp-toluenesulphonate A solution of diphenylmethyl 3 (2'-oxocyclohexyl)methyl 75 (2-thienylacetamido)-ceph-3-em-4-carboxylate (2.25 g., 3.7-5mmole) and pyridine (3.75 ml., 4.75 mmole) in methylene chloride (30ml.) was cooled to l0 and treated with a solution of phosphoruspentachloride (2.35 g., 11.3 mmole) in methylene dichloride (35 ml.)over 5 minutes. The solution was stirred at l0 for 30 minutes. Methanol(37.5 ml.) was added at such a rate that the temperature did not riseabove -10, and when it had all been added, the temperature was allowedto rise to room temperature. After five hours standing the solution wascooled to l0 and treated with water (50 ml), with vigorous stirring.Stirring was continued at room temperature for 30 minutes. The organiclayer was separated and washed succesively with dilute acetic acid,aqueous sodium bicarbonate and Water, and then dried and evaporated. Thegum was dissolved in ethyl acetate (20 ml.) and ether (50 ml.) andtreated with p-toluene-sulphonic acid (0.73 g., 7.7 mmole) in ethylacetate (30 ml.). The title compound separated as colourless prisms(02-80 g., 33%). This material could be recrystallised fromchloroform/ethyl acetate, M.P. 162166 (d), [0:] 0.9 (c. 1, methylenechloride:methanol=4:1 (v./v.)), A (EtOH) .259 nm. (6 6,900), u (Nujol)1780 (B-lactam), 1718 (COOR), and 1700 =O) cm.- N.M.R. (in (CD SO), 7.30(protons in cyclohexanone ring) 7.5- 8.9r (protons in cyclohexanonering). (Found (percent): C, 62.3; H, 5.7; N, 3.9; S, 9.4. C H N O S /2HO requires (percent): C, 62.1; H, 5.7; N, 4.3; S, 9.7.)

(c) 7p-(D-a-amino-u-phenylacetamido) -3-(2'-oxocyclohexyl) methylceph-3em-4-carboxylate A solution of N-(t-butoxycarbonyl)D-phenylglycine (0.92g., 3.35 mmole) in dry tetrahydrofuran (10 ml.) at 6 was treated withtriethylamine (0.54 ml., 3.35 mmole) and then with a solution ofisobutylchloroformate (0.53 g., 3.36 mmole) in dry tetrahydrofuran (4ml.), at

such a rate that the temperature did not rise about 6. After 30 minutesstirring at room temperature the triethylammonium chloride was filteredoff. The filtrate was added to a solution of diphenylmethyl7fl-amino-3-(2'-oxocyclohexyl)methylceph 3-em-4-carboxylate, hydrogen-ptoluenesulphonate (1.5 g., 2.55 mmole) in acetonitrile (8 m1.) andN,N-dimethylacetamide) (4 ml.). After 60 minutes the solvents wereremoved under reduced pressure. A solution of the residue in ethylacetate was washed with aqueous sodium bicarbonate and water and thendried and the solvent evaporated. The gum was treated with anisole (3ml.) and trifluoroacetic acid (12 ml.), and after 5 minutes the reagentsremoved under vacuum. The oil was suspended in water (100 ml.) andtreated with 10%- Amberlite LAl resin -(OAc-") in ether (50 ml.). Aftershaking, the aqueous layer was separated and washed with ethyl acetate(4X 50 ml.) and then freeze-dried to give a white solid 700 mg., 68%),M.P. 150410", (H 261 nm. (e 6,600), v (H 0) 261 nm. 6,600), v (Nujol)1766 (fi-lactam), 1700 =O), 1680 and 1530 (-CONH-), and 1620 -(-COO-)cmr N.M.R. (in (CD SO) 2.52 (phenyl) 7.0-9.0? (protons in cyclohexanering), R, 0.17 (system B), 0.06 (system 0.). Electrophoresis at pH 1.9gives 2 spots, both giving colours with ninhydrin. The faster, whichdoes not absorb ultraviolet light corresponds in this behaviour witha-phenylglycine. The major fraction absorbs U.V. light.

EXAMPLE 21 Sodium 3- (2'-oxocyclohexyl) methyl-7M 2"-thienylacetamido)ceph-3-em-4-carboxylate Diphenylmethyl 3 (2-oxocyclohexyl)methyl-7B-(2"-thienylacetamido)-ceph 3-em-4-carboxylate (1.5 g., 2.5 mmole) wasdissolved in a mixture of anisole (3 ml.) and trifluoroacetic acid (10ml.). After 5 minutes the reagents were removed under reduced pressureand the product precipitated by solution in ethyl acetate and pouringinto petrol. The white solid so obtained was dissolved in ace tone (80ml.) and treated with 10%-sodium ethylhexanoate in acetone (12.5 ml.,7.5 mmole) when the title compound separated as colourless prisms (800mg., 70% 041 +981 (c. 1, H O), x (H 0) 236 nm. (5 13,100), k 260 nm. (65,900), v (Nujol) 1738, {3- lactam), 1690 =O), 1645 and 1532 (CONH-) and1600 (COO cm.- N.M.R. (D 0) 7.0-9.0-r (protons in cyclohexanone ring)(Found (percent): C, 51.4; H, 4.8; N, 5.6; S, 13.4. C H- NaN O S fl/2H Orequires (percent): C, 51.6; H, 4.8; N, 6.0; S, 13.8). R, 0.48 (systemB), 0.61 (system C).

EXAMPLE 22 Diphenylmethyl 3-hydroxymethyl-7B 2'thienylacetceph-3-em-4-carboxylate Diphenylmethyl 3 hydroxymethyl7fi-2'-thienylacetamidoceph-3-ern-4-carboxy1ate (520 mg. 1 mmole) in drytetrahydrofuran (10 ml.) containing pyridine (79 mg., 1 mmole) wastreated dropwise during minutes with a solution of phosphorus tribromide(180 mg., 0.4 mmole) in dry tetrahydrofuran (3 ml.). After 1 hour atroom temperature the mixture was evaporated, the residue shaken withbenzene, and filtered. The filtrate was chromatographed on Kiesel0.05-0.2 (E Merck A.G., Darmstadt, Germany), and the fractions elutedwith benzene:ethy1 acetate=1:8, combined, and evaporated to give a gumwhich, on trituration with cold absolute ethanol, solidified. The solid(180 mg.) was washed with ether and dried in vacuo, 33 (0. 1.0;tetrahydrofuran), x (tetrahydrofuran) 276 nm. (e 9,500), max (bromoform)3390 (NH), 1780 ([Hactam), 1720 (COgCHPhg) and 1500 and 1680 cm. (CONH),NMR (CDC13) 6.2 (CH CONH), 4.20 and 5.07 (protons at the 6- and 27-positions), 5.721- (-CH Br), R, 0.55 (Kieselgel GF 254, ethylacetate:benzene=1:5). (Found (percent): Br, 12.7; S, 11.2. C H BrN O Srequires (percent): C, 55.6; H, 4.0; Br, 13.7; N, 4.8; S, 11.0.)

EXAMPLE 23 (a) Diphenylmethyl 7,8-amino-3isopropoxymethylceph-3-em-4-carboxylate, hydrogen p-toluenesulphonate 3-dichloroacetoxymethyl 78 (2' thieny1acetamido)- ceph 3 em 4 carboxylic acid (14 g., 30.1 mmole)was refluxed in isopropanol (110 ml.) for 45 minutes. After filtrationthrough a pad of Kieselguhr, the isopropanol solution was poured intowater (1000 ml.) and the pH adjusted to 8.5. The solution was extractedwith ethyl acetate (2x100 ml.) and the extracts discarded. The solutionwas then acidified to pH 1.5 with with 2 N-hydrochloric acid andextracted with ethyl acetate (3x100 ml.). The ethyl acetate extractswere dried over magnesium sulphate, and evaporated. The gum wasdissolved in ethyl acetate (40 ml.), and ether (60 m1.) added. Theprecipitate was filtered 01f and the filtrate evaporated to give anorange gum (5.9 g.).

A solution of the above orange gum (5.9 g.) in tetrahydrofuran (65 ml.)was treated with an excess of diphenyldiazomethane (prepared from 4.5 g.of benzophenone hydrazone) in ether ml.). After one hour the solutionwas treated with glacial acetic acid (2 ml.), and evaporated. Theresulting gum was chromatographed on a silicic acid column (4X 15 cms.)with (a) benzene and (b) benzenezethyl acetate=9:1. Those fractionscontaining a substance R ca. 0.7 on thin-layer chromatography (silicagel GF 254, with benzenezethyl acetate=5:l) were combined and evaporatedto give a pale yellow gum (1.73 g.), which was diphenylmethyl3-isopropoxymethyl 75 (2' thienylacetamido)ceph-3-em- 4-carboxylate. The7-sidechain was removed by the general method described in Example 3(a),yielding the title compound (600 mg, 3.3% based on thedichloroacetoxymethyl compound) A (ethanol) 263 nm. (e 6,600), v 1792(fi-lactam), 1728 (COOR) and 1130 ($0 cmr (Found (percent): C, 61.1; H,6.0; N, 4.3; S, 10.3. C H N O- S requires (percent): C, 61.0; H, 5.6; N,4.6; S, 10.5) N.M.R. (CDCl 5.41 and 5.83 (quartet, J =16 Hz, CH OCH(CH6.62, 8.991- (CH CH(CH 2 respectively).

Another experiment afforded diphenylmethyl 3-isopropoxymethyl 7,3aminoceph 3 em 4 carboxylate in a crystalline condition, M.P. 157161(d.), 3 (c. 1.0; ethanol), before the formation of the ptoluenesulphonicacid salt.

(b) 7B-(D-a-amino-wphenylacetamido)-3-isopropoxymethylceph-3-em-4-carboxylic acid Acylation of diphenylmethyl 7fl-amino 3isopropoxymethylceph 3 em 4 carboxylate hydrogen p-toluene sulphonate(0.94 g., 1.47 mmole) with the mixed anhydride from N(t-butoxycarbonyl)-D-phenylg1ycine (0.77 g., 3.06 mmole) andisobutylchloroformate, by the general method described in Example 3(b),gave the title compound (0.46 g., 74%), [(111 -+45 (0. 1, H O), v(water) 260 nm. (5 6,900), v (Nujol) 1766 (B-lactam) and 1695 (COO-)cm." N.M.R. (D 0) 5.26, 6.02, 8.671- (CH OCH(CH respectively) R; 0.76(system A), 0.18 (system B). This material was contaminated byimpurities not revealed on the chromatograms under ultra-violet light;the main impurity is probably a-phenylglycine (ca. 30%). (Found:S:N=1:4.1. Calc. for C H N O S: S:N=1:3.)

Biological results of compounds prepared in examples are given in thefollowing table.

Tube dilution assay (-ylml.)

Mouse protection (EDauJmgJkgJ Gram positive Gram negative dose) Staph.Staph. Staph. Staph. Staph. Stre i S.t1 phi- Pr. mjr- P8. Pilfl- S. au-S. au- Compound of aureus aureus aureus aureua uureus faeca is E. coltmurium abzlzs cyanea reus 663 reus11127 Example No. 604 663 3452 1109211127 850 573 804 431 (S.C.) (S.O.)* HOUR *=subcutaneous.

ROUR**=peroent recovery of the antibiotic from the urine of female ratsfollowing oral administration of the antibiotics.

t -denotes tests carried out on the free acid.

We claim: 1. A compound of the formula 3 u v x (cu ccua T where R iscyclopentyl; phenyl or phenyl substituted by halo, nitro, loweralkanoyloxy, lower alkanoyl, hydroxy, amino, formylamino, lower alkyl orlower alkoxy; and n is or an integer from 1-4; and P is hloro, bromo,iodo, formyloxy, isothiocyanato or haloaeetoxy; or a salt or esterthereof.

2. A compound of the formula where R is cyclopentyl; phenyl or phenylsubstituted by halo, nitro, lower alkanoyloxy, lower alkanoyl, hydroxy,amino, formylamino, lower alkyl or lower alkoxy; n is 0 or an integerfrom 1-4; m is an integer from 1-5 and Z is oxygen or sulphur; and P ischloro, bromo, iodo, formyloxy, isothiocyanoto or haloacetoxy; or a saltor ester thereof.

4. A compound of the formula COOH where R is tat-substitutedarylloweralkanoylamido in which the aryl group is phenyl or phenylsubstituted by halo, hydroxy, lower alkyl, nitro, amino, lower alkanoyl,lower alkoxy or lower alkylmercapto, the a-substituent being hydroxy,carboxy, loweralkoxycarbonyl, cyano, halo, formyloxy, loweralkanoyloxy,lower alkoxy, loweralkanoylamido or amino; and P is chloro, bromo, iodo,formlyoxy, isothiocyanato or haloacetoxy; or a salt or ester thereof. I

6. A compound as defined in claim 5 which is a diphenylmethyl,8,5,5-trichloroethyl or t-butyl ester.

7. A compound as claimed in claim 3 in which n is 0, Z is oxygen, In isl and P is chloro, bromo or iodo.

8. A compound as claimed in claim 5 in which P is chloro, bromo or iodo.

9. A compound as claimed in claim 1 in which R is phenyl, n is 1 and Pis chloro, bromo, or iodo.

10. A compound as claimed in claim 3 in which R is phenyl, n is 0, Z isoxygen, In is 1 and P is chloro, bromo, or iodo.

References Cited UNITED STATES PATENTS 3,322,749 5/1967 Crast 260-443 CNICHOLAS S. RIZZO, Primary Examiner US Cl. X.R. 424246

